Detection and Evaluation of Blast Resistance Genes in Backbone Indica Rice Varieties from South China

Rice blast caused by the pathogenic fungus Magnaporthe oryzae poses a significant threat to rice cultivation. The identification of robust resistance germplasm is crucial for breeding resistant varieties. In this study, we employed functional molecular markers for 10 rice blast resistance genes, namely Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, to assess blast resistance across 91 indica rice backbone varieties in South China. The results showed a spectrum of resistance levels ranging from highly resistant (HR) to highly susceptible (HS), with corresponding frequencies of 0, 19, 40, 27, 5, and 0, respectively. Yearly correlations in blast resistance genes among the 91 key indica rice progenitors revealed Pid2 (60.44%), Pia (50.55%), Pita (45.05%), Pi2 (32.97%), Pikh (4.4%), Pigm (2.2%), Pi9 (2.2%), and Pi1 (1.1%). Significant variations were observed in the distribution frequencies of these 10 resistance genes among these progenitors across different provinces. Furthermore, as the number of aggregated resistance genes increased, parental resistance levels correspondingly improved, though the efficacy of different gene combinations varied significantly. This study provides the initial steps toward strategically distributing varieties of resistant indica rice genotypes across South China.


Introduction
Rice (Oryza sativa L.) production confronts significant threats from diseases and pests, with rice blast, commonly known as "rice cancer", emerging as a major obstacle.The hemibiotrophic fungal pathogen Magnaporthe oryzae triggers rice blast, which is among the most widespread and damaging diseases impacting rice cultivation [1][2][3][4].Given its extensive geographical spread and adaptability to diverse environmental conditions, yield losses attributed to this fungal infection range between 10% and 30% [2,5].This results in the annual devastation of significant rice harvests sufficient to sustain over 60 million individuals and incurs economic losses exceeding $70 billion [6].Recognizing its significant threat to both agricultural productivity and scientific advancement, Science Magazine classified rice blast in 2010 as one of the "most severe bioterrorism threats to food safety" [1].Moreover, the international molecular plant pathology community ranked the rice blast fungus at the forefront of the "top 10 plant pathogenic fungi", underscoring its significance [7,8].In recent years, the incidence rate of rice blast in China has gradually increased, causing increasingly severe damage to rice.Almost all rice-growing areas in China have experienced rice blast, especially in the southern rice-growing areas with a hot and humid climate, which is more conducive to the spread of rice blast [9].
Integrating resistance (R) genes into high-quality rice varieties to develop resistant cultivars has proven to be the most environmentally friendly and sustainable strategy for managing rice blast [4].Over the past decades, researchers have identified over 350 loci associated with quantitative resistance (QRLs) and 100 rice blast-related resistance genes, respectively.Findings revealed consistent resistance levels among the evaluated parental lines over the years, with 59 varieties (64.84%) exhibiting resistance to seedling blight.Across the same evaluation period, resistance levels among the backbone parental lines showed minimal variation, indicating high repeatability of results (Table 1).

Identification and Analysis of Rice Blast Resistance Genes among the Backbone Rice Varieties in South China
This study detected the distribution of 10 rice blast resistance genes, including Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, among the backbone varieties of indica rice in South China (Table 1 and Figure 1).The identification results revealed varied distribution frequencies of the 10 resistance genes among 91 varieties.Pid3 exhibited the highest distribution frequency at 70.33%, followed by Pi5 at 62.64%.Distribution frequencies of Pid2, Pia, and Pita were 60.44%, 50.55%, and 45.05%, respectively.For the remaining genes, distribution frequencies decreased successively: 32.97% for Pi2, 4.4% for Pikh, 2.2% for Pigm, 2.2% for Pi9, and 1.1% for Pi1 (Figure 1).rice in South China (Table 1 and Figure 1).The identification results revealed varied distribution frequencies of the 10 resistance genes among 91 varieties.Pid3 exhibited the highest distribution frequency at 70.33%, followed by Pi5 at 62.64%.Distribution frequencies of Pid2, Pia, and Pita were 60.44%, 50.55%, and 45.05%, respectively.For the remaining genes, distribution frequencies decreased successively: 32.97% for Pi2, 4.4% for Pikh, 2.2% for Pigm, 2.2% for Pi9, and 1.1% for Pi1 (Figure 1).In terms of the number of resistance genes carried, all the varieties of the backbone indica rice carried one or more resistance genes (Table 1).Among them, varieties harboring between two and four resistance genes were 17, 32, and 21, respectively, accounting for 76.92% of the total.Five varieties carried a single resistance gene, accounting for 5.49%.There were 12 varieties with five resistance genes, accounting for 13.19%, and a minimum of 4 varieties carried six resistance genes, accounting for 4.4% (Figure 2a).Further analysis revealed that all six varieties from Sichuan carried a number of resistance genes between two and five, constituting 100% of the cohort, with no detection of varieties carrying only a single or as many as six genes.In Hunan, of the seven cultivated materials, two varieties carried three resistance genes, one carried four, and two carried five, accounting for 28.57%, 14.29%, and 28.57%, respectively.In Zhejiang Province, among 18 varieties, 17 carried between two and five resistance genes, with distribution percentages of 22.22%, 33.33%, 22.22%, and 16.67%, while one variety had a single gene, accounting for 5.56%.In Guangdong Province, all 51 varieties possessed between one and six resistance genes, with 18 varieties carrying three genes, representing the largest subgroup at 35.29%.The number of varieties carrying four resistance genes was the second subgroup, with a total of 15 varieties, accounting for 29.41%.The number of varieties carrying two, five, six, and one resistance genes decreased successively, and the number of In terms of the number of resistance genes carried, all the varieties of the backbone indica rice carried one or more resistance genes (Table 1).Among them, varieties harboring between two and four resistance genes were 17, 32, and 21, respectively, accounting for 76.92% of the total.Five varieties carried a single resistance gene, accounting for 5.49%.There were 12 varieties with five resistance genes, accounting for 13.19%, and a minimum of 4 varieties carried six resistance genes, accounting for 4.4% (Figure 2a).Further analysis revealed that all six varieties from Sichuan carried a number of resistance genes between two and five, constituting 100% of the cohort, with no detection of varieties carrying only a single or as many as six genes.In Hunan, of the seven cultivated materials, two varieties carried three resistance genes, one carried four, and two carried five, accounting for 28.57%, 14.29%, and 28.57%, respectively.In Zhejiang Province, among 18 varieties, 17 carried between two and five resistance genes, with distribution percentages of 22.22%, 33.33%, 22.22%, and 16.67%, while one variety had a single gene, accounting for 5.56%.In Guangdong Province, all 51 varieties possessed between one and six resistance genes, with 18 varieties carrying three genes, representing the largest subgroup at 35.29%.The number of varieties carrying four resistance genes was the second subgroup, with a total of 15 varieties, accounting for 29.41%.The number of varieties carrying two, five, six, and one resistance genes decreased successively, and the number of backbone varieties was seven, six, three, and two, accounting for 13.73%, 11.76%, 5.88%, and 3.92%, respectively (Figure 2b).

Correlation Analysis of Gene Combination Distributions and Resistance Phenotypes
To explore the correlation between the types or number of resistance genes in the tested varieties and their resistance phenotypes to rice blast, we conducted an analysis on the distribution of 10 blast resistance genes in the 91 selected core varieties and their resistance phenotypes to seedling blast.The results showed that among the core varieties

Correlation Analysis of Gene Combination Distributions and Resistance Phenotypes
To explore the correlation between the types or number of resistance genes in the tested varieties and their resistance phenotypes to rice blast, we conducted an analysis on the distribution of 10 blast resistance genes in the 91 selected core varieties and their resistance phenotypes to seedling blast.The results showed that among the core varieties carrying one to six rice blast resistance gene frequencies, 40%, 58.82%, 62.50%, 66.68%, 75%, and 100%, respectively, exhibited disease resistance.With an increase in the number of aggregated resistance genes, the level of the varieties showed an increase accordingly.According to the evaluation of seedling blast resistance over two years, most of the varieties carrying between one and three resistance genes showed a moderate resistance or susceptibility level with a general resistance performance.There were a total of 59 varieties with a seedling blast resistance level of moderate resistance or higher, including 4 from Sichuan, 4 from Hunan, 14 from Zhejiang, 32 from Guangdong, and Hubei, Jiangxi, Anhui, Fujian, and Guangxi all had only 1 variety (Table 2).Further analysis was conducted to evaluate how the presence of 10 distinct resistance genes, Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, affected the frequency of resistance within varieties.The results revealed significant variances in the contribution of each resistance gene to enhancing blast resistance in rice parental lines.Among the variety lines harboring the Pi1 gene, only one exhibited a blast resistance frequency of 0%, while variety lines carrying Pi9, Pigm, and Pikh genes had frequencies of blast resistance at 100%, 100%, and 75%, respectively, involving two, three, and four lines each.Parental lines carrying Pi2 (30 varieties), Pi5 (57 varieties), Pia (46 varieties), Pid2 (55 varieties), Pid3 (64 varieties), and Pita (41 varieties), exhibited blast resistance frequencies of 83.33%, 68.42%, 69.56%, 63.63%, 59.38%, and 73.17%, respectively (Table 2 and Figure 4).Despite the low prevalence of Pi9 and Pigm genes among the tested lines, with frequencies of 2.2% and 3.3%, respectively, these genes showed promising resistance, highlighting their potential utility in rice blast resistance breeding across Hunan, Sichuan, and Anhui.Except for Pi1, varieties carrying Pi2, Pi5, Pia, Pid2, Pid3, Pikh, and Pita demonstrated strong blast resistance performance, suggesting potential utility in rice blast resistance breeding in Southern China.In summary, among the 10 different resistance genes Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, their contributions to resistance breeding were ranked as follows: Pigm, Pi9, Pi2, Pikh, Pita, Pia, Pi5, Pid2, and Pid3.
Accounting for the resistance exhibited by rice varieties against rice blast, an analysis was conducted to evaluate how different combinations of resistance genes contribute to enhancing rice's resistance.Varieties with identical sets of resistance genes were categorized together, resulting in a total of 32 gene combinations.The combination of Pi5+Pia+Pid2+Pid3+Pita was the most prevalent among rice varieties (nine varieties, accounting for 9.9%), exhibiting a rice blast resistance rate of 77.8%, followed closely by the rice varieties carrying the gene combination of Pia+Pid2+Pid3 (seven varieties, accounting for 7.7%), with a rice resistance rate of 42.9%.Rice varieties possessing 20 distinct combinations of resistance genes exhibited moderate to high resistance levels against blast.Among them, the gene combinations of Pi2+Pi5+Pia+Pid2+Pid3+Pita (four varieties, rice blast resis-Plants 2024, 13, 2134 8 of 13 tance rate of 100%), Pi2+Pi5+Pia (three varieties, rice blast resistance rate of 100%), Pi2+Pita (three varieties, rice blast resistance rate of 100%), Pi2+Pid2+Pid3 (two varieties, rice blast resistance rate of 100%), and Pi2+Pi5+Pid2+Pid3 (two varieties, rice blast resistance rate of 100%) emerged as the top five effective combinations of resistance genes (Table 3).This study also found that varieties carrying the same rice blast-resistant gene types exhibited consistent resistance performance, indicating that the resistance gene combination is a key factor determining the level of rice blast resistance in different rice varieties.
Plants 2024, 13, x FOR PEER REVIEW 8 of 13 of 83.33%, 68.42%, 69.56%, 63.63%, 59.38%, and 73.17%, respectively (Table 2 and Figure 4).Despite the low prevalence of Pi9 and Pigm genes among the tested lines, with frequencies of 2.2% and 3.3%, respectively, these genes showed promising resistance, highlighting their potential utility in rice blast resistance breeding across Hunan, Sichuan, and Anhui.Except for Pi1, varieties carrying Pi2, Pi5, Pia, Pid2, Pid3, Pikh, and Pita demonstrated strong blast resistance performance, suggesting potential utility in rice blast resistance breeding in Southern China.In summary, among the 10 different resistance genes Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, their contributions to resistance breeding were ranked as follows: Pigm, Pi9, Pi2, Pikh, Pita, Pia, Pi5, Pid2, and Pid3.Accounting for the resistance exhibited by rice varieties against rice blast, an analysis was conducted to evaluate how different combinations of resistance genes contribute to enhancing rice's resistance.Varieties with identical sets of resistance genes were categorized together, resulting in a total of 32 gene combinations.The combination of Pi5+Pia+Pid2+Pid3+Pita was the most prevalent among rice varieties (nine varieties, accounting for 9.9%), exhibiting a rice blast resistance rate of 77.8%, followed closely by the rice varieties carrying the gene combination of Pia+Pid2+Pid3 (seven varieties, accounting for 7.7%), with a rice resistance rate of 42.9%.Rice varieties possessing 20 distinct combinations of resistance genes exhibited moderate to high resistance levels against blast.Among them, the gene combinations of Pi2+Pi5+Pia+Pid2+Pid3+Pita (four varieties, rice blast resistance rate of 100%), Pi2+Pi5+Pia (three varieties, rice blast resistance rate of 100%), Pi2+Pita (three varieties, rice blast resistance rate of 100%), Pi2+Pid2+Pid3 (two varieties, rice blast resistance rate of 100%), and Pi2+Pi5+Pid2+Pid3 (two varieties, rice blast resistance rate of 100%) emerged as the top five effective combinations of resistance genes (Table 3).This study also found that varieties carrying the same rice blast-resistant gene types exhibited consistent resistance performance, indicating that the resistance gene combination is a key factor determining the level of rice blast resistance in different rice varieties.

Distribution and Manifestation of Blast Resistance Genes in Rice Varieties
Rice blast is a significant threat to rice cultivation, with substantial economic and food security implications.Developing broad-spectrum resistant varieties through gene pyramiding necessitates a comprehensive understanding of the genetic resistance traits in rice varieties.Previous studies have identified functional markers for Pik, Pik-m, Pik-p, Pi1, and Pi64 within China's core germplasm.Pik was found in a single variety (Fidongtang rice), while Pik-p was detected in two varieties (Wenxiangnuo) and Pik-m in two varieties (Cungunuo and Zhengdao 5) [16,30,31].The resistance of rice varieties in the Nanumpet and Mandya regions of Karnataka, India, against rice blast was evaluated.It was found that the single gene frequencies of 20 major blast resistance genes ranged from 10.34% to 100.00%, with Pi9 and Pizt genes showing relatively low and high distribution frequencies, respectively [32].
This study focused on broad-spectrum resistance genes including Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita.The distribution of these genes was analyzed in 91 indica rice backbone varieties using the functional molecular marker technique, and it was found that the Pid3 gene exhibited the highest distribution frequency, reaching 70.33%, followed by the Pi5 gene at 62.64%.The distribution frequencies of the Pid2, Pia, and Pita genes were 60.44%, 50.55%, and 45.05%, respectively.Pid3, Pi5, and Pid2 genes were widely used in rice breeding in Southern China, while Pia, Pita, and Pi2 genes were less distributed.The occurrence rate of Pikh, Pigm, Pi9, and Pi1 genes was notably low.Specifically, Pikh was identified in indica rice backbone varieties, just two from Sichuan and two from Guangdong, while the Pigm gene was found in two indica rice backbone varieties from Sichuan, and the Pi9 gene was observed in two indica rice backbone varieties from Hunan.As for the Pi1 gene, it was detected only in one parent of the indica rice backbone variety originating from Hubei Province.The unbalanced distribution of resistance genes indicated that Pikh, Pigm, Pi9, and Pi1 were not fully utilized in the conventional breeding process of indica rice in South China, and different resistance genes should be combined to improve the resistance of rice varieties [33].
However, this study also revealed that the number of resistance genes does not necessarily correlate with higher resistance levels.For instance, genotypes 23P-81 and 23P-84, despite having multiple genotypes, did not exhibit superior resistance.Similarly, lines with identical resistance genes, such as 23P-36 and 23P-41, showed different resistance performances.These discrepancies suggest that gene interactions, rather than the number of resistance genes, are crucial for effective resistance.Additionally, some materials, like those carrying Pigm and Pikh genes, were limited by small sample sizes, making it challenging to evaluate their resistance performance objectively.This limitation indicates the need for further studies with larger sample sizes to accurately assess the effectiveness of these genes.This study demonstrated that combining resistance genes can expand the resistance spectrum and improve physiological resistance.However, it is essential to select genes with complementary resistance spectra for pyramiding to avoid redundant resistance effects.For example, parental materials 23P-4 and 23P-5 from Sichuan, harboring Pia, Pid2, Pid3, Pigm, and Pikh, exhibited similar resistance levels, while materials 23P-71 and 23P-72 from Guangdong, combining Pi2, Pi5, Pia, Pid2, Pid3, and Pita, showed different results, high lighting the complexity of gene interactions.
Currently, breeding durable and broad-spectrum resistant rice varieties remains a cost-effective strategy for controlling rice blast.This study identified several useful blastresistant genes, including Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, for further utilization.These genes can be targeted for incorporation into major cultivated rice varieties, providing data support for developing new rice varieties resistant to rice blast.Future breeding efforts should aim to integrate the genetic basis of disease resistance with practical field resistance.Combining marker-assisted selection (MAS) and conventional breeding has enabled the incorporation of these broad-spectrum R genes into elite rice varieties, enhancing their resistance and durability against rice blast.

Materials and Strains
The 91 high-quality indica rice varieties tested were from Sichuan, Hubei, Hunan, Jiangxi, Anhui, Fujian, Zhejiang, Guangdong, Guangxi Province, and other regions.All materials were cultivated at the Institute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province.After evaluation of agronomic traits such as plant type, growth duration, rice quality, yield, and rice blast resistance, these lines were deemed suitable for the Zhejiang rice-growing area and selected as breeding backbone varieties for this study.Detailed parental information is shown in Table S1.
Nine physiological strains (ZA63, ZB9, ZC15, ZG1, ZB31, EC13, ZB1, EC9, and EZ15) were selected for rice blast resistance detection.All strains were single-spore isolated from Jinggangshan, Daweishan, Taojiang, and Shanghang in China.Each strain was selected based on pathogenicity tests conducted on physiological races of rice blast in the preceding year.All strains were preserved in the Institute of Crop and Nuclear Technology Utilization of Zhejiang Academy of Agricultural Sciences.

Resistance Identification
Laboratory identification of resistance to rice blast in seedlings.The isolates were isolated from a single spore, and the number of inoculated strains was 9. Previous studies have provided the separation method of blast spores and laboratory identification method [39,40].After soaking the test material with antibiotic 402 for 1 d, it was rinsed with water to promote germination for 1~2 d, sowed in the seedling tray, and 15~20 seeds were sowed for each variety.When the seedling grew to 3 leaves, the test material was sprayed with rice blast strain spore mixed suspension.Twenty-four hours after inoculation, the arch holder was covered with a plastic film to moisturize and a sunshade net was used to prevent direct sunlight.The incidence was investigated 10 d after inoculation according to the International Rice Research Institute (IRRI), grade 0 is highly resistant, grade 1-2 is resistant, grade 3 is moderately resistant, grade 4-5 is moderately sensitive, grade 6-7 is sensitive, and grade 8-9 is highly sensitive [41].

Rice Blast Resistance Gene Detection
Previous studies have informed the choice of these particular genes due to their known association with rice blast resistance in rice or other relevant crops [18][19][20]23,33,34,42].This selection strategy ensures a comprehensive assessment of the genetic factors contributing to rice blast resistance, providing valuable insights for breeding programs and genetic improvement efforts.So 10 mainstream rice blast resistance genes, including Pi1, Pi2, Pi5, Pi9, Pia, Pid2, Pid3, Pigm, Pikh, and Pita, were chosen in this study.DNA was extracted by the conventional CTAB method, and the PCR reaction system was 10 µL at 35 cycles.Molecular-specific labeling primers were provided by Wuhan Jingtai Biotechnology Co., Ltd., Wuhan, China.After the completion of PCR, FAM and HEX were used as the report fluorescence, ROX was used as the reference fluorescence, and the fluorescence signal was read by TECAN infinite F200 enzymoscope (Tecan, Zürich, Switzerland).The fluorescence signal was analyzed and converted to obtain a clear and intuitive typing map, and genotype results were output according to different colors.The specific test was commissioned by Wuhan Jingtai Biotechnology Co., Ltd., Wuhan, China.Markers utilized in this study are detailed in Table S2.

Figure 1 .
Figure 1.Distribution of blast resistance genes among backbone varieties of indica rice.

Figure 1 .
Figure 1.Distribution of blast resistance genes among backbone varieties of indica rice.

Figure 2 .
Figure 2. Percentage and distribution of blast resistance gene combinations among backb eties of indica rice.(a) Percentage of various blast resistance gene combinations.(b) Spat bution frequency of blast resistance combination.Numbers 1-6: The number of blast r genes among different regions.

Figure 2 .
Figure 2. Percentage and distribution of blast resistance gene combinations among backbone varieties of indica rice.(a) Percentage of various blast resistance gene combinations.(b) Spatial distribution frequency of blast resistance combination.Numbers 1-6: The number of blast resistance genes among different regions.Plants 2024, 13, x FOR PEER REVIEW 7 of 13

Figure 3 .
Figure 3. Geographical distribution of rice blast resistance genes across different provinces.

Figure 3 .
Figure 3. Geographical distribution of rice blast resistance genes across different provinces.

Figure 4 .
Figure 4. Correlation analysis of gene type with blast resistance in backbone varieties of indica rice.

Figure 4 .
Figure 4. Correlation analysis of gene type with blast resistance in backbone varieties of indica rice.

Table 1 .
Seedling blast resistance in backbone varieties of indica rice.

Table 2 .
Number of blast resistance genes and resistance evaluation in backbone varieties of indica rice.

Table 3 .
Distribution of blast resistance genes combinations and accordingly resistance levels in backbone varieties of indica rice.